The present invention relates generally to the use of genetic engineering to induce developmental regulation in anther tissue of plants, and more particularly to induce nuclear male sterility, and to genetic sequences useful for same.
Nucleotide and amino acid sequences are referred to herein by sequence identity numbers (SEQ ID NOs) which are defined after the bibliography. A general summary of the SEQ ID NOs is provided before the examples.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Anther-specific genes are those genes that are expressed exclusively in the male reproductive tissues, rather than xe2x80x9chouse-keepingxe2x80x9d genes which are active in all plant cells. Anther-specific genes play an important role in pollen development and, hence, in the control of seed production.
Differentiation and development of the male gametophyte of angiosperms, the pollen grain, depends partly upon transcription of the haploid genome following meiosis (Mascarenhas, 1988). The study of these coordinated events at the molecular level has been considered important in order to understand the developmentally specific regulation and functions of pollen-expressed genes. In this regard, Theerakulpisut et al (1991) studied gene expression in pollen of Brassica campestris. By differential screening of a mature B. campestris pollen cDNA library, an anther-specific clone, designated Bcp1, was isolated.
In work leading up to the present invention, the inventors undertook a detailed investigation of Bcp1 expression with the aim of isolating a genomic clone and to sub-clone and characterise the 5xe2x80x2 upstream regulatory regions of the genomic clone. It has been surprisingly discovered that the genomic clone of Bcp1, i.e. Bgp1, is tissue and developmentally specific thereby providing a means to enable tissue and developmental regulation in plants and in particular to produce nuclear male sterile plants. It has further been discovered that the Bgp1 gene from B. campestris represents a family of homologous genes from a diverse range of plants. By way of shorthand notation, a genomic clone is referred to herein by the genus and/or species of the plant from which it is isolated followed by the term xe2x80x9cBgp1xe2x80x9d. A cDNA clone is referred to in similar fashion except using the term xe2x80x9cBcp1xe2x80x9d.
Accordingly, one aspect of the present invention contemplates a genomic DNA isolate comprising:
(i) all or part of a gene or related genetic sequence preferentially expressed in anther tissue of a plant and substantially not expressed in non-anther tissue; and
(ii) an open reading frame having a nucleotide sequence as set forth in SEQ ID NO. 1:
or having at least 20% similarity to all or part thereof.
The deduced amino acid sequence to the open reading frame defined in SEQ ID NO. 1 is shown in SEQ ID NO. 2.
The expression xe2x80x9cgene or related genetic sequencexe2x80x9d is used in is broadest sense and includes any contiguous series of nucleotides constituting an open reading frame. Generally, an open reading frame comprises at least 48 contiguous nucleotides arranged into triplets without interuption by a stop codon.
A nucleotide sequence having at least 20% similarity to all or a portion of SEQ ID NO. 1 is referred to herein as a xe2x80x9chomologous genexe2x80x9d. Preferably, there is at least 20% similarity to the entire SEQ ID NO. 1 sequence. Even :more preferably, there is at least 30% similarity, still more preferably at least 45% similarity, even still more preferably at least 55-60% similarity, yet even still more preferably at least 75-95% similarity to all or part of SEQ ID NO. 1. A xe2x80x9cpartxe2x80x9d in this context is a contiguous series of at least 20 nucleotides in SEQ ID NO. 1.
Preferably, the genomic DNA isolate is a dicotyledonous plant such as tomato, corn, rice, wheat, raddish, tobacco and oil seed rapes. Particularly preferred plants are Brassica species, Arabidopsis species and Nicotiana species.
In a most preferred embodiment, the plant is Brassica campestris and the genomic DNA isolate has an open reading frame with a sequence as set forth in SEQ ID NO. 1. A preferred homologous gene having at least 20% nucleotide similarity to SEQ ID NO. 1 is from Arabidopsis thaliana comprising an open reading frame with a nucleotide sequence as set forth in SEQ ID NO. 3:
The deduced amino acid sequence of SEQ ID NO. 3 is defined in SEQ ID NO. 4.
Another aspect of the present invention provides a genomic DNA isolate as defined above and further comprising a promoter region 5xe2x80x2 to the open reading frame, wherein said promoter region:
(i) is capable of directing expression in taptum and/or pollen tissue; and
(ii) comprises a nucleotide sequence as set forth in,SEQ ID NO. 5:
or having at least 20% similarity to all or part thereof.
Preferred promoters comprise the promoter defined in SEQ ID NO. 5 and the promoter defined in SEQ D NO. 6 which has the following nucleotide sequence:
Yet another aspect of the present invention relates to a genomic DNA isolate comprising:
(i) all or part of a gene or related genetic sequence preferentially expressed in anther tissue of a plant and substantially not expressed in non-anther tissue;
(ii) a promoter region capable of directing expression in tapetum and/or pollen tissue;
(iii) a nucleotide sequence substantially as set forth in SEQ ID NO. 7:
or having at least 20% similarity to all or part thereof.
In a preferred embodiment, the above genomic DNA isolate further comprises:
(iv) a nucleotide sequence which is capable of hybridising under low stringency conditions to all or part of a nucleotide sequence substantially complementary to SEQ ID NO. 7.
Most preferred genomic DNA isolates comprise SEQ ID NO. 7 and SEQ ID NO. 8, the latter which has the following nucleotide sequence:
Still yet another aspect of the present invention contemplates an isolated nucleic acid molecule which is capable of hybridising under low stringency conditions to the genomic DNA isolates defined above. Preferred nucleic acid molecules comprise a complementary strand of all or part of SEQ ID NO. 1 or SEQ ID NO. 3. A xe2x80x9cpartxe2x80x9d in this context includes an oligonucleotide.
A further aspect of the present invention provides a genetic construct comprising:
(i) a promoter region capable of directing expression of a nucleotide sequence when operably linked downstream thereof in tapetum and/or pollen tissue; and
(ii) said promoter being capable of hybridising under low stringency conditions to a complementary strand of SEQ ID NO. 5.
For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al., Supra at pp 387-389 which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1-0.5% w/v SDS at 37-45xc2x0 C. for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25%-0.5% w/v SDS at xe2x89xa745xc2x0 C. for 2-3 hours or high stringent conditions as disclosed by Sambrook et al., Supra.
In a further related embodiment, there is provided a nucleic acid isolate having a sequence of nucleotides comprising or a complementary sequence of nucleotides comprising SEQ ID NO. 5 or a promoter functional derivative, fragment, part, homologue or analogue thereof. The latter functional derivative and like molecules comprise at least 20% nucleotide sequence similarity to SEQ ID NO. 5. An example of a promoter having at least 20% nucleotide similarity to SEQ ID NO. 5 is the promoter from A. thaliana Bgp1 having the sequence set forth in SEQ ID NO. 6.
In accordance with these and other aspects of the present invention, the term xe2x80x9cpromoterxe2x80x9d is used in its most general sense and refers to any nucleotide sequence which binds RNA polymerase and directs same to a transcriptional start site whereupon a gene or other nucleotide sequence downstream of said promoter is transcribed. A nucleotide sequence xe2x80x9cdownstreamxe2x80x9d of the promoter is also said to be xe2x80x9crelativexe2x80x9d the promoter.
The term xe2x80x9cgenetic constructxe2x80x9d is used in its most broadest sense to include an isolated nucleic acid molecule comprising a sequence of nucleotides.
Preferably, the promoter is from a Brassica species such as B. compestris or from an Arabidopsis species such as A. thaliana. Preferably, the genetic construct is transformable and operable in dicotyledon plants and in particular a Brassica species, Arabidopsis species or a Nicotiana species.
The genetic construct may be conveniently engineered so as to place an endonuclease restriction site in a region 3xe2x80x2 of the promoter to thereby readily enable the insertion of nucleotide sequences downstream of the promoter for their transcription. Generally, the inserted restriction site is unique to the genetic construct or may be represented twice but separated by a length of nucleic acid to be deleted upon restriction digestion of the genetic construct and followed by insertion of the required nucleotide sequence to be transcribed.
The genetic construct of the present invention may comprise solely the promoter and optionally a nucleotide sequence downstream thereof or, alternatively, may comprise additional nucleotide sequences constituting promoter regulatory region(s), transcribed sequence regulatory regions, a marker (eg. antibiotic resistance, chemical compound resistance or enzyme), autonomous replication region and/or genome integration sequence. The promoter may be the naturally occurring promoter or may be an active fragment or part thereof or a derivative, analogue or homologue of the promoter.
By xe2x80x9cderivativexe2x80x9d is meant to include any single or multiple nucleotide deletion, insertion and/or substitution to the promoter nucleotide sequence, provided said derivative is still active in tapetum and/or pollen tissue. Manipulation of the nucleotide sequence at known predetermined sites or random mutagenesis are conveniently accomplished by any number of techniques including M13, transposon and/or oligonucleotide mutagenesis. Various techniques are described by Maniatis et al (1989).
Homologues and analogues of the promoter include promoters having a nucleotide sequence having at least 20%, preferably at least 30% similarity, more preferably at least 45% similarity, still more preferably at least 55-60% similarity and even more preferably at least 75-95% similarity to the first mentioned promoter and which function in anther tissue.
Most preferred promoters comprise the sequence SEQ ID NO. 5 or SEQ ID NO. 6.
The promoter of the present invention is tissue specific for anther tissue. More particularly, the promoter is specific for tapetum and/or pollen tissue. However, this is not intended to exclude genetic constructs based on the promoter of the present invention but modified to be capable of expression in non-anther tissues.
The nucleotide sequence down stream of the promoter might give rise to antisense RNA or may encode specific traits such as a xe2x80x9clethal genexe2x80x9d or a xe2x80x9cMiller genexe2x80x9d to specifically render a pollen grain infertile or incapable of maturation. The nucleotide sequence may also encode a trait, for example, which renders the pollen grain more resistant to predator or pathogen attack. In one particular embodiment, the nucleotide sequence downstream of the promoter is a ribozyme capable of targetting a mRNA transcript corresponding to SEQ ID NO. 1 or SEQ ID NO. 3 or a homologous genetic sequence thereof.
According to this latter embodiment, there is provided a ribozyme which comprises a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least part of a target mRNA sequence transcribed from a genomic Bgp1 gene as hereinbefore defined wherein the catalytic region is capable of cleaving said target mRNA thereby substantially down regulating expression of said genomic DNA isolate. A ribozyme according to this aspect of the invention may also be a polyribozyme.
Methods for the construction of ribozyme are conveniently disclosed in Haseloff and Gerlach (1988) and in International Patent Application No. WO89/05852. Preferably, the ribozyme is under the control of a Bgp1 promoter as hereinbefore described.
The present invention further extends to a hybrid genetic sequence comprising a ribozyme as hereinbefore described fused, linked or otherwise chemically bonded to one or more sequence of nucleotides which is/are substantially antisense to all or part of SEQ ID NO. 1 or a homologous sequence (e.g. antisense to all or part of SEQ ID NO. 3).
The antisense sequence may flank both sides of a ribozyme or may be located to one end of said ribozyme. Reference to a ribozyme in this context includes reference to a polyribozyme. A xe2x80x9csubstantially antisensexe2x80x9d molecule is a molecule capable of hybridising under physiological conditions to the reference sequence (e.g. SEQ ID NO. 1 or SEQ ID NO. 3) to a sufficient extent to reduce translation of said target sequence into functional protein or which results in male sterility.
The present invention is particularly exemplified using the promoter isolated from a genomic clone of Bcp1, the genomic clone being designated herein xe2x80x9cBgp1xe2x80x9d, from Brassica species or non-Brassica species with similar acting promoters. Such other promoters are referred to herein as xe2x80x9chomologous promotersxe2x80x9d and include the promoter from the homologous gene A. thaliana Bgp1 defined by SEQ ID NO. 6. Most of the Bgp1 promoter is required for pollen expression and in particular nucleotide regions xe2x88x92580 to xe2x88x92767, xe2x88x92322 to xe2x88x92580 and xe2x88x92116 to xe2x88x92168 whereas the nucleotide region up to xe2x88x92116 is only required for tapetum expression.
According to a preferred embodiment, the present invention provides an isolated nucleic acid molecule carrying a promoter capable of directing expression in tapetum and pollen tissue and comprising the following nucleotide sequence identified as SEQ ID NO. 5, including functional derivatives or homologue having at least 20% nucleotide similarity to all or a part thereof and/or which are capable of hybridising to a complementary strand thereof under at least low stringency conditions.
According to another embodiment, there is provided an isolated nucleic acid molecule carrying a promoter capable of directing expression in pollen tissue but not tapetum, said nucleic acid molecule comprising the following nucleotide sequence identified as SEQ ID NO. 9:
including functional derivatives or homologues having at least 20% nucleotide similarity to all or a part thereof and/or which are capable of hybridising to a complementary strand thereof under at least low stringency conditions.
A particularly important homologue is SEQ ID NO. 6 from A. thaliana. 
Preferably, the nucleotide sequence of SEQ ID NO. 5, SEQ ID NO. 9 or SEQ ID NO. 6 is modified by the introduction of a restriction endonuclease cleavage site to facilitate the insertion of an operably linked second nucleotide sequence downstream of the promoter.
Preferably, the nucleotide sequence of the present invention form part of a vector.
The identification of a tissue and developmentally dependent promoter enables the production of genetic constructs which can be used to generate transgenic plants having certain traits expressed or down regulated. For example, the function of the Bgp1 gene can be conveniently disrupted using antisense RNA or a ribozyme. Conveniently, the cDNA clone Bcp1 is inserted in the reverse orientation relative the Bgp1 promoter. This construct, when introduced into a suitable host, produces antisense RNA which disrupts expression of the Bgp1 gene. Although not intending to limit the present invention to any one theory of mode of action, it is possible the antisense RNA forms a duplex with Bgp1 RNA to thereby prevent its translation. Transgenic plants carrying the particular construct are generally male sterile but female fertile.
According to this aspect of the present invention there is provided an antisense construct:
(i) which comprises a nucleic acid molecule comprising at least eight contiguous nucleotides;
(ii) which is capable of hybridising under physiological conditions to all or part of SEQ ID NO. 1 or a homologous.-sequence thereof; and
(iii) which, in use, is capable of down regulating expression of a plant Bgp1 gene.
In this context, a xe2x80x9chomologousxe2x80x9d sequence comprises a nucleotide sequence having at least 20% similarity to all or part of SEQ ID NO. 1 and which is a Bgp1 gene.
Preferably, the antisense construct is at least 20 nucleotides long. More preferably, the antisense construct is at least 50-100 nucleotides long. Even more preferably, the antisense construct is all or part of a plant Bcp1 or Bgp1 in reverse orientation relative a promoter.
The term xe2x80x9cdown regulatesxe2x80x9d or similar expressions such as xe2x80x9cdown regulatingxe2x80x9d means a reduction in the amount of full length Bgp1 mRNA as determined by hybridisation or extent of translation into a Bgp1 product or, most conveniently, generation of substantially male sterile plants.
Yet another aspect of the present invention contemplates a method for generating male sterile plants, said method comprising transforming a cell or group of cells of said plant with a genetic construct capable of directing expression of a nucleotide sequence having a deleterious effect on tapetum and/or pollen tissue, regenerating a transgenic plant from said transformed cells and growing and/or maintaining said transgenic plant under conditions to thereby having a deleterious effect on said tapetum and/or pollen tissue resulting in said plant being substantially male sterile.
In an alternative embodiment, there is provided a method for generating male sterile plants, said method comprising introducing into a cell or group of cells of said plant, a genetic construct comprising all or part of a Bgp1, said Bgp1 having a nucleotide sequence substantially similar to an endogenous Bgp1 of the plant and then regenerating a plant from said cells. This method is term xe2x80x9cco-suppressionxe2x80x9d. The introduced Bgp1 may be with or without a promoter. By xe2x80x9csubstantiallyxe2x80x9d similar is meant an exogenous Bgp1 comprising 85-100% nucleotide sequence similarity to an endogenous Bgp1.
Still yet another aspect of the present invention provides a transgenic male sterile plants such as those made by the above method.